BACKGROUND. RNAi is a powerful genetic tool for dissecting functional vulnerabilities in cancer cells. Current generation of siRNA libraries are not knockdown validated and can lead to a high false discovery rate. PURPOSE. In this project we aim to accomplish the following goals: 1) constructing a high quality, knockdown validated siRNA library that target Ras family and Ras effector genes; 2) using this siRNA library to screen a panel of human cancer cell lines with or without KRAS mutations to identify those siRNA and siRNA combinations that show selective lethality among the KRAS mutant cell lines; and 3) functional dissection of the mechanisms of candidate KRAS synthetic lethal interactions using molecular and pharmacological tools. SIGNIFICANT MATERIALS AND METHODS. Knockdown validated siRNA library targeting Ras pathway genes. ACCOMPLISHMENT. We have identified and validated a set of siRNAs against Ras pathway genes. We demonstrated that these siRNAs can be combined to knockdown multiple genes. The first phase of this study has been published. We are currently systematically evaluating siRNA combinations for their selective lethality in KRAS mutant cancer cell lines.